GR is a nuclear receptor (NR), a family of intracellular ligand-regulated transcription factors (McMaster, A. et al., Exp Physiol 92:299-309 (2007)). GR is expressed ubiquitously in humans. Activation of GR by hormones such as cortisol causes nuclear translocation, interaction with co-regulators, and binding to specific genomic sites to regulate transcription (Yamamoto, K. R. et al., Cold Spring Harbor Symposia on Quantitative Biology 63:587-598 (1998); So, A. Y. et al., PLoS Genet 3:e94 (2007)). This mediates the broad systemic effects of GR signaling and underlies glucocorticoid treatment of diverse immune-mediated diseases such as asthma and rheumatoid arthritis. However, severe dose-limiting side effects occur, including osteoporosis, muscle wasting, and diabetes (Schacke, H. et al., Pharmacology & Therapeutics 96:23-43 (2002)). No existing drugs selectively induce beneficial effects of GR.
Beneficial and harmful effects of glucocorticoids are due to selective activation or repression of particular genes by GR. This selectivity is in part based on tissue-specific factors and cross-talk pathways (Kassel, O. et al., Molecular and Cellular Endocrinology 275:13-29 (2007)). For example, GR reduces the expression of certain inflammatory cytokines by inhibiting other transcription factors such as AP-1 and NF-Kb (Smoak, K. A. et al., Mech Ageing Dev 125:697-706 (2004)). Conversely, GR increases expression of RANKL, a gene co-regulated by the Vitamin D receptor that activates bone resorption by osteoclasts (Hofbauer, L. C. et al., Endocrinology 140:4382-4389 (1999); Kim, S. et al., Mol. Cell. Biol. 26:6469-6486 (2006)). Controlling GR activity at certain tissues or promoters has profound therapeutic implications.
Efforts to achieve this goal have focused primarily on developing selective GR ligands that simply induce a subset of GR activities (Rosen, J. et al., Endocr Rev 26:452-464 (2005); Cole, T. J. et al., Medicinal Chemistry 3:494-506 (2007); Honer, C. et al., Mol Pharmacol 63:1012-1020 (2003)); see also assays described in U.S. Pat. No. 5,968,738 and WO/2001/516077. However, it is not yet possible to predict GR gene regulation based on ligand design, and it remains uncertain whether new ligands can produce therapeutically relevant transcriptional selectivity. Moreover, transcription based screens for novel GR modulators generally measure GR activation at a single experimental promoter (Fan, F. et al., ASSAY and Drug Development Technologies 5:127-136 (2007)). This does not allow efficient identification of molecules that produce promoter-specific responses. Accordingly, although numerous GR agonists with different potencies and/or modes of delivery are in clinical use, dose equivalency generally results in similar clinical responses and side effects (Adams, N. et al., Cochrane Database Syst Rev, CD002310 (2007)). Non-ligand modulation of GR is an alternative strategy to achieve the desired transcriptional output, potentially enabling tissue or promoter-specific GR effects.
To address this problem, we developed a high throughput system to measure GR activity at four promoters. This permits discovery of genes or molecules that alter GR signaling in a promoter-specific fashion. We have applied this system to identify selective GR modulators in an initial screen of 1040 natural products and FDA-approved compounds.
In addition, the assay disclosed herein can be applied to detect selective gene regulation by ligand dependent transcription factors such as nuclear receptors, by assaying up to five different receptors in a single well. The assay can be used to detect general modulators, cell specific modulators (e.g., a drug that blocks a promoter in one cell type but not another, or protein specific modulators (e.g., a drug that causes a single protein to turn on only a subset of genes that it would ordinarily activate). The assay can help speed the identification of novel ligands or compounds that work through non-receptor binding sites (i.e., cross talk pathways). For example, this assay could be used to identify modulators of glucocorticoid signaling that would cause the receptor to activate immunosuppression genes while avoiding induction of genes that cause muscle wasting.
Additional potential targets include the androgen receptor, which is responsible for initiation and progression of prostate cancer, hirsutism, and mediates muscle hypertrophy in response to anabolic steroids; the estrogen receptor, which is a key target for breast cancer, heart disease, and the female reproductive tract; the thyroid hormone receptor, which plays a key role in regulating metabolism and bone loss; and the mineralocorticoid receptor, which regulates salt metabolism. The general class of nuclear receptors includes the retinoic acid receptor, RXR, the liver X receptor, and peroxisome proliferator activated receptor (PPAR), which plays a key role in fat metabolism. All mediate their effects via selective regulation of gene expression, and have similar modes of action, and thus could be used in this system.